Kurzfassung: Abstract 3811 Background: A large number of genes have been found mutated in CMML including 18 encoding signaling molecules (CBL, N/KRAS, JAK2, FLT3, KIT), epigenetic regulators (TET2, IDH1/2, DNMT3A, ASXL1, EZH2) transcription (RUNX1, NPM1) and splicing (SRSF2, SF3B1, U2AF1, ZRSR2) factors. We report the genotypic patterns, clinical correlates and prognostic impact of mutations in those 18 genes in a large cohort of CMML patients (pts). Methods: Bone marrow or peripheral CD14+ cells from 224 CMML pts from a non interventional study (n=186) or a phase II decitabine trial (n=38; Braun Blood 2011) were genotyped by mutation specific techniques and Sanger sequencing for up to 18 genes (depending on available material): TET2, IDH1, IDH2, DNMT3A, CBL, NRAS, KRAS, JAK2V617F, FLT3, KIT, NPM1, RUNX1, ASXL1, EZH2, SF3B1, SRSF2, U2AF1 and ZRSR2. The number of TET2 alleles with a functional Cystein Rich (CysR) domain (Delhommeau NEJM 2009) was predicted based on mutation type and zygosity, assuming that double mutations affect independent alleles. Overall (OS) and AML-free (AMLFS) survival were analyzed from the date of genotyping. Results: 224 CMML pts (152M/72F, median age 75y) were genotyped at diagnosis (37%) or after a median of 7.2 months of evolution (none had received hypomethylating agents [HMA] before genotyping); WHO diagnosis was CMML-1/2 in 78%/22%, 70% pts had normal karyotype, 22% had extramedullary disease (EMD); 13 pts had autoimmune manifestations (AIM). The most frequently mutated genes were TET2 (58%), SRSF2 (47%) and ASXL1 (38%). Mutations in RUNX1, CBL and NRAS were found in 14%, 11% and 10% of pts, respectively (resp). All other genes were mutated in 〈 10% of pts. Only 5% pts lacked any mutation, and 70% had ≥2 mutated genes; TET2, IDH1 and IDH2 mutations, present in 64% of pts, were mutually exclusive. Mutations in splice and signaling genes were present in 63% and 35% of pts, with 2 mutated genes within each group in 3% and 2% pts, resp. ASXL1 mutations were less frequent in the presence of TET2 mutations (P 〈 .0001). Significant mutual associations included ASXL1/RUNX1, ASXL1/NRAS, TET2/SRSF2, RUNX1/SRSF2 and U2AF1/IDH2. In multivariate analysis accounting for those interactions, TET2 status was the only independent predictor of hemoglobin values (median 10.2 vs 11.9 g/dL in wildtype [wt] vs mutated pts, P 〈 .0001) with a gene dosage effect (P=.0003). Platelet counts were higher in JAK2V617F pts, and lower in pts with RUNX1, TET2 or SRSF2 mutations. WBC and monocyte counts were higher in pts with ASXL1 and NRAS mutations. EMD was associated to ASXL1, CBL, KRAS and JAK2 mutations. EMD, CMML-2 and abnormal karyotype were less frequent in TET2 mutated pts. All 13 pts with AIM had at least one mutated gene, with no specific genotype spectrum. With a median follow-up of 25.4 months, median OS and AMLFS were 32.2 and 28.0 months resp. In univariate analysis, OS was decreased in pts with IDH2 mutations (P=.04), and AMLFS was shorter in pts with NRAS (P=.04), RUNX1 (P=.03) and SRSF2 (P=.04) mutations. ASXL1 mutations markedly reduced OS (median 18.5 vs 35.7 months in wt pts) and AMLFS (median 12.5 vs 34.7 months, both P 〈 .0001), with similar results in the 74 pts who received HMA during follow-up. In the 224 pts, there was no significant effect of overall TET2 status (wt vs mutated) on OS or AMLFS (both P=.09) but median OS was 29.3, 35.7 months and not reached in pts with 2 (57%), 1 (30%) and 0 (13%) TET2 alleles with a putatively functional CysR domain (P=.01). Similar differences were noted for AMLFS (P=.008). In multivariate analysis including peripheral blood counts, WHO classification, cytogenetics, disease evolution and therapy, ASXL1 was the only gene whose mutations independently predicted inferior OS (HR: 2.44, 95% CI: 1.36–4.37, P=.003)and AMLFS (HR: 2.54, 95% CI: 1.46–4.42, P=.001); SRSF2 mutations only predicted inferior AMLFS (HR: 2.05, 95% CI: 1.15–3.65, P=.02). The total number of mutated genes as a continuous variable, which was higher in ASXL1 mutated pts (mean 3.0 vs 1.8, P 〈 .0001), was the only genetic variable to retain prognostic value when added to those models (OS and AMLFS both P 〈 .0001). Conclusion: TET2, SRSF2 and ASXL1 are the most frequent mutated genes in CMML. The number and location of TET2 mutations may impact CMML presentation and outcome. The total number of mutated genes has the strongest prognostic relevance, but ASXL1 mutational status provides a robust surrogate prognostic marker for daily practice. Disclosures: No relevant conflicts of interest to declare.

Kurzfassung: Introduction A relative increase in classical monocyte fraction (cMo, CD14++CD16-) over 94% of total peripheral blood monocytes, as measured by flow cytometry, distinguishes a CMML from a reactive monocytosis with a 94.1% specificity and a 92.8% sensitivity (Selimoglu-Buet, 2015; Talati, 2017; Patnaik, 2017; Selimoglu-Buet, 2017; Hudson, 2018). This phenotype is independent of the absolute monocyte count, WHO subtype of CMML, its dysplastic (MD) vs proliferative (MP) feature and its mutational background. The limitation of this diagnostic tool is the changes in monocyte subset repartition induced by an associated inflammatory disease that increase the fraction of intermediate monocytes (iMo, CD14++CD16+), leading to underestimation of cMo accumulation (Selimoglu-Buet, 2017). The present study explores the prognostic significance of cMo accumulation in CMML. Patients and methods Among CMML patients (diagnosis according to 2016 WHO criteria) included in our previous studies, we selected patients diagnosed from June 2012 to March 2017 in four centers in which follow-up was actualized in June 2019. Disease was classified according to WHO 2016 (CMML-0, -1, -2 and MP vs MD subtypes). When possible, the CMML Prognostic Scoring System (CPSS) was calculated. Peripheral blood flow cytometry data were reanalyzed in a blind fashion by a skilled operator. Inflammatory CMML were defined by a cMo percentage 〈 94% associated with a bulbous aspect on flow cytometry profile (Figure 1A). The absolute cMo count in the peripheral blood was determined by combining flow cytometry measurement of cMo fraction and the total monocyte count provided by Complete Blood Count. Results One hundred and twenty-nine CMML patients (mean age: 75±10 years, sex ratio: 2.2) were classified into 53 CMML-0 (41%), 58 CMML-1 (45%) and 18 CMML-2 (14%) with 97 dysplastic forms (75%). Eight patients (6%) having received DNA damaging therapies (radiations or chemotherapies) were considered as secondary CMML. Eleven patients (9%) had evolved from a previous myelodysplastic syndrome. The median white blood count, hemoglobin level and platelet count were 9 G/l (IQR: 5.9-12.9), 11.8 g/dl (IQR: 9.7-13.1), and 121 G/l (IQR: 73-212), respectively. The CPSS score, calculated in 119 cases, classified the patients into "Low" (n=55), "Intermediate-1" (n=33), "Intermediate-2 (n=29) and "High" (n=2) categories. Twenty of the 114 patients (18%) with available follow-up (median duration: 26.7 months [13.9-36.5]) had received hypomethylating agents and 3 (3%) were allografted. A cMo percentage above the threshold (97.0% [IQR: 96.3-98.1]) was detected in 112 patients (87%) whereas 17 (13%), referred thereafter as "inflammatory CMML", displayed cMo 〈 94% (91.6% [IQR: 89.9-93.2], p 〈 0.001) with an inflammatory state (C-reactive protein: 8 mg/L [IQR: 3.1-20.2] (Figure 1B). Importantly, the absolute count of cMo was similar in inflammatory (1.6 G/l [IQR: 1.1- 1.8] ) and classical (1.7 G/l [IQR: 1.2- 2.6], p=0.25) CMML (Figure 1C). Focusing on total monocyte and cMo absolute counts, we did not detect any significant difference between CMML-0, CMML-1 and CMML-2. While cMo percentages were similar in MP and MD subtypes, the total monocyte (6.0 ±6.0 G/l vs 1.7 ±0.7 G/L, p 〈 0.001) and the cMo (5.8 ±5.7 G/L vs 1.6 ±0.7 G/L, p 〈 0.001) counts were increased in MP compared to MD CMML. A poor prognosis CPSS score at diagnosis also correlated with an increase in the absolute cMo count ("Intermediate-2 or High": 4.5 ±6.0 G/L vs "Low or Intermediate-1": 2.0 ±1.7 G/L; p 〈 0.001) (Figure 1D). A Receiver Operator Curve (ROC) defined a cut-off value of 2.0 G/L to identify patients with poorest outcome. Indeed, CMML patients with an absolute cMO count ≥2.0 G/L at diagnosis had a higher risk to progress to acute myeloid leukemia (AML) (HR=0.34 [95% CI, 0.12 - 0.91]) (Figure 1E). Eventually, patients with an absolute cMO count ≥2.0 G/L at diagnosis had poorer overall survival (OS) compared with patients with a lower absolute cMO count (HR=0.42 [95% CI, 0.22 - 0.80] ; median OS: 31.9 months vs not reached, p=0.009) (Figure 1F). Conclusion These results demonstrate that in CMML patients, regardless of the relative cMo percentage, a high absolute count of circulating cMo at diagnosis ≥2.0 G/L correlates with a poor outcome, including a higher risk of AML progression and shorter survival. Figure 1 Disclosures Fenaux: Jazz: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Aprea: Research Funding. Wagner Ballon:Alexion: Consultancy, Honoraria.

Kurzfassung: Context. The perhaps only CMML-specific Randomized Clinical Trial (RCT) established hydroxyurea (HY) as the main treatment (Tx) for advanced proliferative CMML (Wattel Blood 1996). In Europe, the only hypomethylating agent (HMA) approved in CMML is AZA in non proliferative CMML-2. Phase 2 trials reported the activity of decitabine (DAC) in advanced proliferative CMML (Braun Blood 2011, Santini Leukemia 2018). We performed a RCT of DAC (±HY during the first 3 cycles) vs HY alone in those pts. Methods. The DACOTA trial (EudraCT 2014-000200-10) accrued pts with previously untreated (or & lt; 6 weeks of HY), proliferative (WBC ≥ 13x109/L) CMML with advanced disease defined per Wattel et al as presence of extramedullary disease or ≥2 criteria among: BM blasts ≥5%, abnormal karyotype (except -Y), ANC ≥ 16x109/L, Hb & lt; 10 g/dL, platelets & lt; 100 x109/L or splenomegaly & gt; 5 cm below costal margin. Pts were randomized 1:1 to DAC (20 mg/m2/d IV 5d/28d) or HY (1g/d, adjusted on WBC, 28d cycles) and treated until death, AML transformation or progression. The primary endpoint was EFS, events being death, transformation to AML, progression of myeloproliferation after 3+ cycles or progression of blasts and cytopenias after 6+ cycles. Response was assessed with IWG 2006 criteria modified to account for improvement of myeloproliferation, after central morphology review. Intent-to-treat analyses were done considering missing responses as failures. Results. From Oct 2014 to Sep 2019, 217 pts from 47 centers were screened and 170 randomized (84 DAC and 86 HY), including 12 pts (6 DAC and 6 HY) who never started Tx. Median age was 73 years (IQR 68-78). WHO was CMML-NA/1/2 in 2, 114 and 54 pts, respectively (resp). Median WBC 34.9 x109/L (IQR 22.9-55.7). Cytogenetic risk (Such Haematologica 2011) was fav 69%, int 12%, adv 18% NA 1%. Mutations in TET2, SRSF2, ASXL1 and signaling genes (CBL, JAK2, FLT3, KIT, NRAS, KRAS and CSF3R) were present in 64%, 51%, 62% and 57% resp. 72 pts had received HY for a median 27 days prior to randomization. Aside from older age in the HY arm (median 74 vs 71.5y in the DAC arm), there was no imbalance between Tx arms. DAC and HY pts received a median of 5 (IQR 3-12) and 6 (IQR 3-14) cycles, resp. As of 15th June 2020, 5 and 10 DAC and HY pts were still on Tx. Reasons for Tx cessation in the DAC arm were death (n=19), AML transformation (n=16), progression (n=9) , hematological toxicity (n=13) or other (n=21). Reasons for Tx cessation in the HY arm were death (n=14), AML transformation (n=13), progression (n=18), hematological toxicity (n=6) or other (n=20). 126 and 85 pts received 3 and 6 cycles, resp. In the ITT population, ORR at 3 cycles was 56% (7CR, 25 mCR±HI, 15 SD+HI) and 30% (0 CR, 8 mCR±HI, 18 SD+HI) in the DAC and HY arms, resp (p=0.0011) and ORR at 6 cycles was 32% (6 CR, 9 mCR±HI, 12 SD+HI) and 17% (2 CR, 4 mCR±HI, 9 SD+HI) in the DAC and HY arms, resp (p=0.033). Median response duration was 15.9 vs 18.2 months (mos) in the DAC and HY arm, resp (p=0.81). Infection and hemorrhage occurred at least once in 49% and 31% of pts, resp. 55% of DAC pts and 38% of HY pts required hospitalization at least once (p=0.05). Non-heme ≥ grade 2 AEs occurred in 79% and 63% of DAC and HY arms, resp (p=0.03). Grade ≥3 cardiac AEs occurred in 13 DAC and 4 HY pts, resp. With a median follow-up of 13.9 mos, median EFS was 12.6 vs 10.3 mos in the DAC and HY arms, resp (reference DAC arm, HR= 1.14 CI95 0.8-1.64, p= 0.46). Median AML-free survival (AMLFS) was 13.6 and 15.8 mos in the DAC and HY arms resp (p=0.86). Median OS was 18.4 and 23.1 mos in the DAC and HY arms, resp (p=0.72). Considering death and AML transformation as competing risks there was no significant difference in cumulative incidence of AML (p=0.1) or death without transformation (p=0.06) between arms. 30 pts from the HY arm received an HMA (DAC n= 13, AZA n= 16, both=1) after study exit. Censoring at HMA onset in the HY arm, median OS was 18.4 vs 30.4 in the DAC and HY arm, resp (p=0.15). 13 pts were transplanted (DAC n= 10, HY n= 3). There was no interaction between Tx arm and CMML-0/1 vs -2, platelets ≥ vs & lt;100 x109/L and anemia (Hb & lt; 8 g/dL or RBC-TD vs Hb ≥8) on both EFS and OS (all p & gt;0.05). Conclusion. RCTs are feasible in advanced proliferative CMML, which remains an unmet medical need. In these pts, DAC did not provide an overall or event-free survival advantage over HY. HY remains a valid option in advanced proliferative CMML. However, one third of HY pts subsequently received an HMA and more DAC pts achieved a response and were bridged to HSCT. Figure Disclosures Itzykson: Abbvie: Honoraria; Daiichi Sankyo: Honoraria; Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Sanofi: Honoraria; BMS (Celgene): Honoraria; Janssen: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Stemline: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncoethix (now Merck): Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Santini:BMS, J & J, Novartis: Honoraria; Acceleron, BMS, Menarini, Novartis: Consultancy; Takeda, Pfizer: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding. Lionel:Abbvie: Consultancy; Takeda: Consultancy; Celgene/BMS: Consultancy, Research Funding; Novartis: Consultancy; Jazz: Consultancy, Research Funding. Thepot:astellas: Honoraria; novartis: Honoraria; sanofi: Honoraria; celgene: Honoraria. Giagounidis:AMGEN: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Luebbert:Janssen: Research Funding. Park:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Other: Travel expenses. Stamatoulas Bastard:Pfizer: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Celgene: Honoraria; Takeda: Consultancy. Solary:Janssen: Research Funding. Platzbecker:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria; Amgen: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Geron: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Fenaux:Novartis: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Jazz: Honoraria, Research Funding. OffLabel Disclosure: Decitabine for CMML with WBC & gt; 13 x109/L.

Kurzfassung: Hydroxyurea (HY) is a reference treatment of advanced myeloproliferative neoplasms. We conducted a randomized phase III trial comparing decitabine (DAC) and HY in advanced myeloproliferative chronic myelomonocytic leukemias (CMML). PATIENTS AND METHODS Newly diagnosed myeloproliferative CMML patients with advanced disease were randomly assigned 1:1 to intravenous DAC (20 mg/m 2 /d days 1-5) or HY (1-4 g/d) in 28-day cycles. The primary end point was event-free survival (EFS), events being death and acute myelomonocytic leukemia (AML) transformation or progression. RESULTS One-hundred seventy patients received DAC (n = 84) or HY (n = 86). Median age was 72 and 74 years, and median WBC count 32.5 × 10 9 /L and 31.2 × 10 9 /L in the DAC and HY arms, respectively. Thirty-three percent of DAC and 31% of HY patients had CMML-2. Patients received a median of five DAC and six HY cycles. With a median follow-up of 17.5 months, median EFS was 12.1 months in the DAC arm and 10.3 months in the HY arm (hazard ratio [HR], 0.83; 95% CI, 0.59 to 1.16; P = .27). There was no significant interaction between treatment effect and blast or platelet count, anemia, CMML Prognostic Scoring System, Groupe Francophone des Myelodysplasies, or CMML Prognostic Scoring System–mol risk. Fifty-three (63%) DAC patients achieved a response compared with 30 (35%) HY patients ( P = .0004). Median duration of response was similar in both arms (DAC, 16.3 months; HY, 17.4 months; P = .90). Median overall survival was 18.4 months in the DAC arm and 21.9 months in the HY arm ( P = .67). Compared with HY, DAC significantly reduced the risk of CMML progression or transformation to acute myelomonocytic leukemia (cause-specific HR, 0.62; 95% CI, 0.41 to 0.94; P = .005) at the expense of death without progression or transformation (cause-specific HR, 1.55; 95% CI, 0.82 to 2.9; P = .04). CONCLUSION Compared with HY, frontline treatment with DAC did not improve EFS in patients with advanced myeloproliferative CMML (ClinicalTrials.gov identifier: NCT02214407 ).

Kurzfassung: Several prognostic scoring systems have been proposed for chronic myelomonocytic leukemia (CMML), a disease in which some gene mutations—including ASXL1—have been associated with poor prognosis in univariable analyses. We developed and validated a prognostic score for overall survival (OS) based on mutational status and standard clinical variables. Patients and Methods We genotyped ASXL1 and up to 18 other genes including epigenetic (TET2, EZH2, IDH1, IDH2, DNMT3A), splicing (SF3B1, SRSF2, ZRSF2, U2AF1), transcription (RUNX1, NPM1, TP53), and signaling (NRAS, KRAS, CBL, JAK2, FLT3) regulators in 312 patients with CMML. Genotypes and clinical variables were included in a multivariable Cox model of OS validated by bootstrapping. A scoring system was developed using regression coefficients from this model. Results ASXL1 mutations (P 〈 .0001) and, to a lesser extent, SRSF2 (P = .03), CBL (P = .003), and IDH2 (P = .03) mutations predicted inferior OS in univariable analysis. The retained independent prognostic factors included ASXL1 mutations, age older than 65 years, WBC count greater than 15 ×10 9 /L, platelet count less than 100 ×10 9 /L, and anemia (hemoglobin 〈 10 g/dL in female patients, 〈 11g/dL in male patients). The resulting five-parameter prognostic score delineated three groups of patients with median OS not reached, 38.5 months, and 14.4 months, respectively (P 〈 .0001), and was validated in an independent cohort of 165 patients (P 〈 .0001). Conclusion A new prognostic score including ASXL1 status, age, hemoglobin, WBC, and platelet counts defines three groups of CMML patients with distinct outcomes. Based on concordance analysis, this score appears more discriminative than those based solely on clinical parameters.

Kurzfassung: Abstract 3997 We have shown previously that cells identified as monocytes in the peripheral blood of patients with chronic myelomonocytic leukemia (CMLL) included a variable proportion of CD14-negative, CD24-positive immature granulocytes. These cells synthesize and secrete alpha-defensin 1–3 that inhibit M-CSF-driven monocyte differentiation into macrophages through interaction with the P2Y6 purinergic receptor (Droin N et al, Blood 2010). In the present study, we show that these CD14-,CD24+ immature granulocytes also inhibit the proliferation of autologous lymphocytes activated with anti-CD3 and anti-CD28 antibodies through cell-cell contact. This functional property suggested that these cells could be “myeloid-derived suppressive cells” (MDSC), which was supported by their phenotype that included expression of CD15 marker at their surface and survivin, S100A8 and S100A9, Cyclin D2 and Cyclin D3 at the mRNA level. STAT3 and STAT6 were found constitutively phosphorylated in these immature granulocytes that responded to Toll-like receptor agonists such as LPS or Pam-6. CD14-positive monocytes of the leukemic clone activated these MDSC through production of IL-13 and induction of arginase 1 mRNA, which could be reproduced by recombinant IL-13. On the other hand, activation of these MDSC did not require induction of the nitric oxide synthase mRNA, in agreement with their granulocytic origin. We were able to generate immature myeloid cells expressing CD24 with morphology similar to that of peripheral blood MDSC by in vitro culture of various subpopulations of bone marrow CD34-positive cells obtained from CMML patients, including the most immature CD34+/CD38-/CD90+ cells. Furthermore, generation of these cells could be recapitulated in vivo by xenotransplantation of CMML CD34+ cells in NOG mice, albeit with lower efficacy than CD14+ cells. In patients with high grade CMML included in a phase II clinical trial, decitabine was observed to decrease both CD14+,CD24- monocytes and CD14-,CD24+ immature granulocytes. Altogether, these data suggest that CMML initiating cells generate CD14-positive monocytes and, in most patients, an additional population of CD14-negative immature granulocytes with suppressive properties towards innate and acquired immune response. Generation of these cells may account for the high sensitivity of CMML patients to autoimmune and infectious diseases. Disclosures: Fenaux: Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding; ROCHE: Honoraria, Research Funding; AMGEN: Honoraria, Research Funding; GSK: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Cephalon: Honoraria, Research Funding.

Kurzfassung: The cytidine analogues azacytidine and 5-aza-2’-deoxycytidine (decitabine) are commonly used to treat myelodysplastic syndromes, with or without a myeloproliferative component. It remains unclear whether the response to these hypomethylating agents results from a cytotoxic or an epigenetic effect. In this study, we address this question in chronic myelomonocytic leukaemia. We describe a comprehensive analysis of the mutational landscape of these tumours, combining whole-exome and whole-genome sequencing. We identify an average of 14±5 somatic mutations in coding sequences of sorted monocyte DNA and the signatures of three mutational processes. Serial sequencing demonstrates that the response to hypomethylating agents is associated with changes in DNA methylation and gene expression, without any decrease in the mutation allele burden, nor prevention of new genetic alteration occurence. Our findings indicate that cytosine analogues restore a balanced haematopoiesis without decreasing the size of the mutated clone, arguing for a predominantly epigenetic effect.

Kurzfassung: Non-classical monocyte subsets may derive from classical monocyte differentiation and the proportion of each subset is tightly controlled. Deregulation of this repartition is observed in diverse human diseases, including chronic myelomonocytic leukemia (CMML) in which non-classical monocyte numbers are significantly decreased relative to healthy controls. Here, we identify a down-regulation of hsa-miR-150 through methylation of a lineage-specific promoter in CMML monocytes. Mir150 knock-out mice demonstrate a cell-autonomous defect in non-classical monocytes. Our pulldown experiments point to Ten-Eleven-Translocation-3 (TET3) mRNA as a hsa-miR-150 target in classical human monocytes. We show that Tet3 knockout mice generate an increased number of non-classical monocytes. Our results identify the miR-150/TET3 axis as being involved in the generation of non-classical monocytes.

Kurzfassung: Despite their location at the cell surface, several receptor tyrosine kinases (RTK) are also found in the nucleus, as either intracellular domains or full length proteins. However, their potential nuclear functions remain poorly understood. Here we find that a fraction of full length Colony Stimulating Factor-1 Receptor (CSF-1R), an RTK involved in monocyte/macrophage generation, migrates to the nucleus upon CSF-1 stimulation in human primary monocytes. Chromatin-immunoprecipitation identifies the preferential recruitment of CSF-1R to intergenic regions, where it co-localizes with H3K4me1 and interacts with the transcription factor EGR1. When monocytes are differentiated into macrophages with CSF-1, CSF-1R is redirected to transcription starting sites, colocalizes with H3K4me3, and interacts with ELK and YY1 transcription factors. CSF-1R expression and chromatin recruitment is modulated by small molecule CSF-1R inhibitors and altered in monocytes from chronic myelomonocytic leukemia patients. Unraveling this dynamic non-canonical CSF-1R function suggests new avenues to explore the poorly understood functions of this receptor and its ligands.